Diet-induced obesity (DIO) in rodents is accompanied by heightened inflammatory signaling through the NF-?B pathway in the hypothalamus. Growing evidence suggests that during refined high-fat diet (HFD) feeding, this hypothalamic response favors weight gain by reducing leptin and insulin sensitivity. For example, interventions that reduce hypothalamic NF-?B signaling attenuate DIO, but the mechanisms linking hypothalamic NF-?B signaling to DIO remain to be defined. Recent evidence indicates that DIO is accompanied by increased hypothalamic production of the reactive oxygen radical superoxide, and a concomitant increase in the expression of the superoxide-generating enzyme NADPH oxidase 2 (NOX2). NOX2 is expressed in microglia, the macrophage of the brain, and generates extracellular superoxide when these cells are activated. A role for microglia to limit hypothalamic inflammation is suggested by our preliminary data and by evidence that mice lacking factors necessary for the activation of myeloid cells, including microglia, are more susceptible to DIO than littermate controls. Microglia have a well-documented ability to restrain inflammatory signaling in other models of neuronal injury, and our preliminary evidence indicates that NOX2-mull mice gain more fat mass that controls when fed a HFD but not unrefined chow. Here, we investigate the hypothesis that during HFD feeding, microglial superoxide generation limits hypothalamic leptin resistance and associated fat gain by activating a neuroprotective phenotype in microglia. To this end, we will characterize the metabolic phenotype of NOX2-null mice, and investigate the consequences of NOX2 deficiency for both inflammation in the mediobasal hypothalamus (MBH) and the associated microglial response during HFD feeding. In complementary studies, we will inject PEGylated recombinant superoxide dismutase and catalase into the MBH of genetically intact animals to establish a causal role of superoxide in DIO resistance. We anticipate that this intervention will increase fat gain in rats fed HFD. We will also investigate whether during HFD feeding hypothalamic microglia increase superoxide production relative controls fed standard chow, and whether this increase depends on the presence of NOX2. Together, these studies will expand our understanding of mechanisms underlying DIO, and thereby inform the development of more effective strategies for the prevention and treatment of human obesity. PUBLIC HEALTH RELEVANCE: Feeding rodents refined high-fat diets causes inflammatory changes in the hypothalamus that contribute to the rapid accumulation of excess body fat. Although inflammatory signaling is implicated in the pathogenesis of obesity in this setting, its cause is unknown. Here we investigate the role played by the reactive oxygen radical superoxide in the hypothalamic inflammation and associated weight gain induced by high-fat diet feeding. Our recent finding that mice lacking the superoxide-generating enzyme NADPH oxidase 2 (NOX2) have increased susceptibility to fat gain when fed high-fat diets suggests that NOX2-derived superoxide, generated by local microglia (the macrophage of the brain) limits both weight gain and hypothalamic inflammation in this setting. The goal of this proposal is to identify the cellular source of excess superoxide in the hypothalamus of rats fed a high-fat diet and determine the role of NOX2 and superoxide in diet-induced obesity.